Enhancement of plasmid DNA yields during fed-batch culture of a fruR-knockout Escherichia coli strain
Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be o...
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Published in | Biotechnology and applied biochemistry Vol. 52; no. Pt 1; p. 53 |
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Main Authors | , , |
Format | Journal Article |
Language | English |
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United States
01.01.2009
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Abstract | Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be optimized to attain higher plasmid yields. To date, nearly all production optimization efforts are focused on media or fermentation process design. Although there has been a simple empirical evaluation of the available host strains, there is a lack of systematic effort at engineering these host strains for improved plasmid DNA production. In view of this, we engineered DH5alpha WT (wild-type) cells carrying a DNA vaccine plasmid by knocking out the fruR (fructose repressor) [also known as the Cra (catabolite repressor activator)] global regulator gene and evaluated the growth and plasmid yields of these P+ (plasmid-bearing) fruR cells (fruR-knockout cells) during fed-batch cultures with exponential feeding. The P+ fruR cells showed a more rapid accumulation of plasmid DNA towards the end of the fed-batch cultures compared with the P+ WT cells. As a result, the specific plasmid yield of the P+ fruR cells was 21% higher than that of the P+ WT cells [19.2 versus 15.9 mg/g DCW (dry cell weight)]. These results demonstrate that, from an initial high-yielding fermentation process, the knockout of the fruR global regulator gene in E. coli DH5alpha further improves plasmid yields during fed-batch culture. |
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AbstractList | Well-characterized derivatives of Escherichia coli K12 such as DH5alpha are the host strains commonly used for plasmid DNA production. Owing to the prospective clinical demand for large quantities of plasmid DNA for gene therapy and DNA vaccination, existing plasmid production processes need to be optimized to attain higher plasmid yields. To date, nearly all production optimization efforts are focused on media or fermentation process design. Although there has been a simple empirical evaluation of the available host strains, there is a lack of systematic effort at engineering these host strains for improved plasmid DNA production. In view of this, we engineered DH5alpha WT (wild-type) cells carrying a DNA vaccine plasmid by knocking out the fruR (fructose repressor) [also known as the Cra (catabolite repressor activator)] global regulator gene and evaluated the growth and plasmid yields of these P+ (plasmid-bearing) fruR cells (fruR-knockout cells) during fed-batch cultures with exponential feeding. The P+ fruR cells showed a more rapid accumulation of plasmid DNA towards the end of the fed-batch cultures compared with the P+ WT cells. As a result, the specific plasmid yield of the P+ fruR cells was 21% higher than that of the P+ WT cells [19.2 versus 15.9 mg/g DCW (dry cell weight)]. These results demonstrate that, from an initial high-yielding fermentation process, the knockout of the fruR global regulator gene in E. coli DH5alpha further improves plasmid yields during fed-batch culture. |
Author | Ow, Dave Siak-Wei Oh, Steve Kah-Weng Yap, Miranda Gek-Sim |
Author_xml | – sequence: 1 givenname: Dave Siak-Wei surname: Ow fullname: Ow, Dave Siak-Wei email: ow@bti.a-star.edu.sg organization: Bioprocessing Technology Institute, 20 Biopolis Way, 06-01 Centros, Singapore. dave ow@bti.a-star.edu.sg – sequence: 2 givenname: Miranda Gek-Sim surname: Yap fullname: Yap, Miranda Gek-Sim – sequence: 3 givenname: Steve Kah-Weng surname: Oh fullname: Oh, Steve Kah-Weng |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/18380624$$D View this record in MEDLINE/PubMed |
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CitedBy_id | crossref_primary_10_1007_s00253_012_4308_5 crossref_primary_10_1016_j_biotechadv_2011_12_005 crossref_primary_10_1016_j_jbiotec_2014_06_008 crossref_primary_10_1080_10826068_2016_1141302 crossref_primary_10_1002_jctb_5011 crossref_primary_10_1088_1755_1315_948_1_012071 crossref_primary_10_1186_1475_2859_10_52 crossref_primary_10_1016_j_biotechadv_2009_02_003 crossref_primary_10_1016_j_vaccine_2009_10_057 crossref_primary_10_1002_biot_201100062 crossref_primary_10_1007_s00253_009_1889_8 |
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SubjectTerms | Electrophoresis, Agar Gel Escherichia coli - genetics Escherichia coli - growth & development Escherichia coli Proteins - genetics Gene Knockout Techniques - methods Plasmids - biosynthesis Repressor Proteins - genetics Spectrometry, Fluorescence |
Title | Enhancement of plasmid DNA yields during fed-batch culture of a fruR-knockout Escherichia coli strain |
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